1. Field of the Invention
The present invention relates to proteins and nucleic acids related to salt tolerance in plants.
2. Description of the Background
In Arabidopsis thaliana, the Salt Overly Sensitive 2 (SOS2) gene is required for intracellular Na+ and K+ homeostasis. Mutations in SOS2 cause Na+ and K+ imbalance and render plants more sensitive toward growth inhibition by high Na+ and low K+ environments. We isolated the SOS2 gene through positional cloning. SOS2 is predicted to encode a serine/threonine type protein kinase with an N-terminal catalytic domain similar to that of the yeast SNF1 kinase. Sequence analyses of sos2 mutant alleles reveal that both the N-terminal catalytic domain and the C-terminal regulatory domain of SOS2 are functionally essential. The steady-state level of SOS2 transcript is up-regulated by salt stress in the root. Autophosphorylation assays show that SOS2 is an active protein kinase. In the recessive sos2-5 allele, a conserved glycine residue in the kinase catalytic domain is changed to glutamate. This mutation abolishes SOS2 autophosphorylation, indicating that SOS2 protein kinase activity is required for salt tolerance.
Control of intracellular ion homeostasis is essential for all cellular organisms. Most cells maintain relatively high K+ and low Na+ concentrations in the cytosol. In plants. this is achieved through coordinated regulation of transporters for H+, K+, and Na+. At the plasma membrane, a family of P-type H+-ATPases serves as the primary pump that generates a protonmotive force driving the active transport of other solutes, including K+ and Na+ (1). Several plant K+ channels and transporters have been molecularly characterized. The inward rectifying K+ channel AKT1 is essential for root K+ uptake in Arabidopsis thaliana (2, 3). Expression characteristics indicate that the KAT1 channel is involved in K+ influx in Arabidopsis guard cells (4, 5). Recently, an outward rectifying K+ channel has been shown to be essential for unloading K+ into the Arabidopsis root xylem (6). The wheat HKT1 gene product functions as a high-affinity K+ transporter (7). In addition, a family of KUP genes exists in Arabidopsis. At least one of them, KUP1, encodes a protein that can function as a dual-affinity K+ transporter (8, 9). Na+ enters plant cells passively, presumably through K+ transport systems (10), Unlike animals or fungi. plants do not seem to possess Na+/K+-ATPases or Na+-ATPases. Na+ efflux s achieved through the activities of Na+/H+ antiporters on the plasma membrane. Much of the Na+ that enters the cell is compartmentalized into the vacuole through the action of vacuolar Na+/H+ antiporters (11, 12). The driving force for the vacuolar transporters is the protonmotive force created by vacuolar V-type H+-ATPases and the H+-pyrophosphatase (1, 13). Although there has been great progress in the characterization of K+ and Na+ transporters in plants, little is currently known about their regulation.
In the trophic chain, plant roots play pivotal roles by taking up mineral nutrients from soil solutions. Plant roots experience constant fluctuations in soil environments. A frequent variant in the soil solution is Na+ concentration (14). Na+ is not an essential ion for most plants. In fact, the growth of the majority of plants, glycophytes, is inhibited by the presence of high concentrations of soil Na+. External Na+ causes K+ deficiency by inhibiting K+ uptake into plant cells (15). Na+ accumulation within the cell is toxic to many cytosolic enzymes. In contrast, many cellular enzymes are activated by K+, which is the most abundant cation in the cytoplasm. Certain cytoplasmic enzymes arc especially prone to Na+ inhibition when K+ concentration is reduced (16). Therefore, maintaining intracellular K+ and homeostasis to preserve a high K+/Na+ ratio is important for all cells and especially critical for plant cells.
A family of Arabidopsis sos (salt overly sensitive) mutants defective in the regulation of intracellular Na+ and K+ homeostasis was recently characterized (15, 17, 18). The sos mutants are specifically hypersensitive to inhibition by high concentrations of external Na+ or Lixe2x88x92 (17, 18). In response to high Na+ challenge, the sos2 and sos3 mutants accumulate more Na+ and retain less K+ than wild-type plants (18). The mutants are also unable to grow when the external K+ concentration is very low (17, 18). These phenotypes suggest that the mutant plants are defective in the regulation of K+ and Na+ transport (18). The SOS3 gene was recently cloned and shown to encode an EF hand-type calcium-binding protein that shares significant sequence similarities with animal neuronal calcium sensors and the yeast calcineurin B subunit (19). In yeast, calcineurin is a central component in the signaling pathway that regulates Na+ and Kxe2x88x92 homeostasis (20, 21). Loss-of-function mutations in calcineurin B cause increased sensitivity of yeast cells to Na+ or Li+ stress.
Because of limited water supplies and the widespread use of irrigation, the soils of many cultivated areas have become increasingly salinized. In particular, modern agricultural practices such as irrigation impart increasing salt concentrations when the available irrigation water evaporates and leaves previously dissolved salts behind. As a result, the development of salt tolerant cultivars of agronomically important crops has become important in many parts of the world. For example, in salty soil found in areas such as Southern California, Arizona, New Mexico and Texas.
Dissolved salts in the soil increase the osmotic pressure of the solution in the soil and tend to decrease the rate at which water from the soil will enter the roots. If the solution in the soil becomes too saturated with dissolved salts, the water may actually be withdrawn from the plant roots. Thus the plants slowly starve though the supply of water and dissolved nutrients may be more than ample. Also, elements such as sodium are known to be toxic to plants when they are taken up by the plants.
Salt tolerant plants can facilitate use of marginal areas for crop production, or allow a wider range of sources of irrigation water. Traditional plant breeding methods have, thus far, not yielded substantial improvements in salt tolerance and growth of crop plants. In addition, such methods require long term selection and testing before new cultivars can be identified.
Accordingly, there is a need to increase salt tolerance in plants, particularly those plants which are advantageously useful as agricultural crops.
We report here the positional cloning of the SOS2 locus. SOS2 is predicted to encode a serine/threonine type protein kinase with an N-terminal catalytic domain highly similar to those of yeast SNF1 and mammalian AMPK kinases. Sequence analyses of several sos2 mutant alleles point to a functional requirement of both the N-terminal catalytic domain and the C-terminal regulatory domain of SOS2. SOS2 is expressed in both the root and shoot. In the root, SOS2 mRNA is up-regulated by salt stress. Autophosphorylation assays demonstrate that SOS2 is an active protein kinase. Furthermore, a mutation that abolishes SOS2 autophosphorylation renders plants hypersensitive to salt stress, indicating that SOS2 protein kinase activity is necessary for salt tolerance. This demonstrates that a protein kinase is essential for intracellular Na+ and K+ homeostasis and plant salt tolerance.
Thus, the present invention provides an isolated polynucleotide which encodes a protein comprising the amino acid sequence in SEQ ID NO:2.
In a preferred embodiment the polypeptide has serine/threonine kinase activity.
In another preferred embodiment the polynucleotide comprises SEQ ID NO:1, polynucleotides which are complimentary to SEQ ID NO:1, polynucleotides which are at least 70%, 80% and 90% identical to SEQ ID NO:1; or those sequence which hybridize under stringent conditions to SEQ ID NO:1, the stringent conditions comprise washing in 5 X SSC at a temperature from 50 to 68xc2x0 C.
In another preferred embodiment the polynucleotides of the present invention are in a vector and/or a host cell. Preferably, the polynucleotides are in a plant cell or transgenic plant. Preferably, the plant is Arabidopsis thaliania or selected from the group consisting of wheat, corn, peanut cotton, oat, and soybean plant. In a preferred embodiment, the polynucleotides are operably linked to a promoter, preferably an inducible promoter.
In another preferred embodiment the present invention provides, a process for screening for polynucleotides which encode a protein having serine/threonine kinase activity comprising hybridizing the polynucleotide of the invention to the polynucleotide to be screened; expressing the polynucleotide to produce a protein; and detecting the presence or absence of serine/threonine kinase activity in said protein.
In another preferred embodiment, the present invention provides a method for detecting a nucleic acid with at least 70% homology to nucleotide SEQ ID NO:1, sequences which are complimentary to SEQ ID NO:1 and/or which encode a protein having the amino acid sequence in SEQ ID NO:2 comprising contacting a nucleic acid sample with a probe or primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of claim 1, or at least 15 consecutive nucleotides of the complement thereof.
In another preferred embodiment, the present invention provides a method for producing a nucleic acid with at least 70% homology to the polynucleotides of the present invention comprising contacting a nucleic acid sample with a primer comprising at least 15 consecutive nucleotides of the nucleotide sequence of claim 3, or at least 15 consecutive nucleotides of the complement thereof.
In another preferred embodiment, the present invention provides a method for making SOS2 protein, comprising culturing the host cell carrying the polynucleotides of the invention for a time and under conditions suitable for expression of SOS2, and collecting the SOS2 protein.
In another preferred embodiment, the present invention provides a method of making a transgenic plant comprising introducing the polynucleotides of the invention into the plant.
In another preferred embodiment, the present invention provides method of increasing the salt tolerance of a plant in need thereof, comprising introducing the polynucleotides of the invention into said plant.
In another preferred embodiment, the present invention provides an isolated polypeptide comprising the amino acid sequence in SEQ ID NO:2 or those proteins that are at least 70%, preferably 80%, preferably 90% and preferably 95% identity to SEQ ID NO:2. Preferably, the polypeptides have serine/therenine kinase activity.